Myxobacteria are soil dwelling Gram-negative bacteria. They survive by secreting a variety of hydrolytic enzymes that break down the organic matter as well as other living microorganisms in their environment. They are most noted for their ability to form fruiting body structures when they are starved for nutrients (Dworkin, 1996, “Recent advances in the social and developmental biology of the myxobacteria” Microbiol Rev 60:70–102). These fruiting bodies house thousands of dormant myxospores that are resistant to a variety of environmental stresses. Within the last decade they have gained prominence as producers of secondary metabolites, some of which are currently being exploited as potential drug candidates (Reichenbach, 2001, “Myxobacteria, producers of novel bioactive substances” J Industrial Microbiology and Biotechnology 27:149–156). Analysis of myxobacteria reveals that bacterial of the genus Sorangium are a rich source of unique bioactive secondary metabolites (Reichenbach, 2001; Reichenbach and Höfle, 1999, “Myxobacteria as producers of secondary metabolites,” p. 149–179, in Grabley and Thiericke, ed., Drug Discovery from Nature. Springer Verlag, Berlin; and Reichenbach and Höfle, 1993, Production of bioactive secondary metabolites, p. 347–397, in M. Dworkin and D. Kaiser, ed., Myxobacteria II. American Society for Microbiology, Washington, D.C.), the most prominent of which are the epothilones (Altmann, 2001, “Microtubule-stabilizing agents: a growing class of important anticancer drugs” Curr Opin Chem Biol 5:424–31). Biosynthesis of epothilones remains the method of choice for obtaining commercially useful quantities of these compounds.
However, Sorangium strains are some of the most difficult myxobacteria with which to work. They have the longest doubling time of myxobacteria, up to 16 hours, and very few genetic tools are available. S. cellulosum is difficult to engineer, due to the low efficiency of introducing DNA into the bacteria (Jaoua et al., 1992, “Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for their integration into the chromosome” Plasmid 28:157–65) and the limited number of molecular tools and markers that have been developed to date. For example, a genetic transformation system based on homologous recombination has been described (see U.S. Pat. No. 5,686,295), but this system appears to work inefficiently, if at all, in most instances. Thus, introducing exogenous DNA for expression or to make knockout mutations, particularly when using a vector containing a small region of homology, is problematic.
The ability to make mutations in Sorangium would be extremely useful to identify the gene clusters responsible for the synthesis of secondary metabolites; a single strain of Sorangium can produce several different known secondary metabolites (for example, So ce12 makes four known compounds; see Reichenbach and Höfle, 1999), and in addition, may harbor gene clusters that synthesize compounds that have not been identified. Many of the secondary metabolites isolated from myxobacteria are complex polyketides synthesized by type I polyketide synthases (PKS), which are large multimodular proteins (For review, see Hopwood et al., 1990 “Molecular genetics of polyketides and its comparison to fatty acid biosynthesis” Annu Rev Genet 24:37–66; Khosla et al., 1999, “Tolerance and specificity of polyketide synthases” Annu Rev Biochem 68:219–53; and Shen, B., 2003, “Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms” Curr Opin Chem Biol 7:285–95). A method for making mutations in Sorangium to correlate which of several polyketide synthase gene clusters in a genome is responsible for synthesizing which polyketide would be valuable. In addition, technology has been developed to manipulate a PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide, or to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters (see McDaniel, R., et al, 2000; Weissman, K. J. et al. 2001; McDaniel, et al., 1993; Xue, et al., 1999; Ziermann, et al., 2000; U.S. Pat. Nos. 6,033,883 and 6,177,262; and PCT publication Nos. 00/63361 and 00/24907).
Thus, methods and reagents for making mutations in Sorangium would be a valuable tool, simplifying correlation of polyketide synthase gene clusters and specific polyketides, modifying polyketide synthase gene clusters, and having many other uses.
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